Human cytomegalovirus is a widespread pathogen that is the leading viral cause of birth defects and a major cause of morbidity and mortality in immunocompromised adults. Its interaction with an infected host is complex. It can undergo latency in some cell types and actively replicate to produce infectious progeny in a variety of other cell types. Given its prominence as a human pathogen, it is essential that we continue to advance our understanding of the molecular mechanisms that underlie human cytomegalovirus replication and pathogenesis. The long-term objective of my research program is to elucidate the function of human cytomegalovirus genes that regulate the interaction of the virus with its host cell and thereby control the processes of viral replication and pathogenesis. Here I propose to first perform a global mutational analysis of both an HCMV laboratory strain (AD169) and a clinical strain (FIX) and then to use the libraries to explore the functions of viral genes and the contribution of individual viral genes to host cell tropism. My specific aims include: (1) Perform growth analysis of a library of mutant HCMV AD 169 variants carrying transposon insertions; (2) Employ the library of transposon mutants in AD 169 as the starting point for the detailed analysis of specific gene functions; and (3) Prepare and analyze a library of mutant HCMV FIX variants, focusing on the requirement for function of the mutated ORFs during viral replication in human fibroblasts, endothelial cells, macrophages and smooth muscle cells.